The present invention relates to clinical assay processes utilizing denaturation (conversion of endogenous material to an assayable form) and more particularly to processes for determination of human sera and cell levels of vitamin B.sub.12 and/or folate by radioassay without use of boiling for denaturation. The invention may also be applied to other target components (analytes) of sera or cells (endogenous content) and to other forms of assay.
Measurement of the concentration of folate and vitamin B.sub.12 in the serum and of folate in red blood cells, is useful as an adjunct in the clinical diagnosis and differentation of various types of nutritional anemia. Deficiencies in these vitamins predominantly arise from either inadequate diet, malabsorption, or alcoholism.
Deficiency in vitamin B.sub.12 may result in neurological damage. Further, as this vitamin is necessary to transport N-methyltetrahydrofolate across human cell walls, its absence may also disturb folate metabolism and result in megaloblastic anemias. As megaloblastosis may also be produced by folate deficiency due to other causes, it is necessary to determine if it is caused by a deficiency of either or both vitamins.
Before the advent of radioassay procedures, microbiological assays were the principal means of determining the levels of serum folate and vitamin B.sub.12. Such assays are time consuming and are subject to errors from the other factors that may influence growth rate such as presence of antibiotics and antimetabolites in the sample.
Following the development of the general features of saturation analysis or competitive protein binding (CPB) radioassay, and of specific radioassays for folate and vitamin B.sub.12, microbiological assays have been largely replaced. The radioassay techniques not only produced results from correlated well with microbiological values, but also often provided better precision and freedom from interferences at lower costs of time and money. The principle of competitive binding used in these radioassays involves binding of analytes and tagged analogs thereof to a fixed quantity of a material, generally a protein, that has binding sites that are specific for the analytes and analogs. For vitamin B.sub.12, purified intrinsic factor (e.g. using hog intrinsic factor, human IF, chicken or toad fish sera) is used as a binding protein; for folate assays, the folate binding protein in .beta.-lactoglobulin is used. The competition for a limited number of binding sites of the specific protein occurs between the analyte present in the sample or standard and a small fixed quantity of a tracer (an analog of the analyte that has been labeled with a radioisotope). When there is zero endogenous analyte present, the tracer has no competition and occupies all of the sites of the binding protein. Separation of the bound and unbound (free) fractions of tracer, followed by measurement of one of these fractions permits one to construct a standard curve of the amount of radioactivity as a function of the concentration of analyte. Amounts of analyte in unknown samples can be read from the standard curve using radioactivity readings of such samples.
It is an important object of the present invention to provide improved assay processes of the class described.